Stem cells are “primitive” cells devoted to maintain functional and structural integrity of tissues, by replacement of damaged mature cells. Stem cells can be distinguished depending on their different ability to differentiate into different kinds of tissues (different degree of “potency”).
Stem cells are distributed in all tissues, and are particularly available from sources like bone marrow, dental pulp, adipose tissue, peripheral blood, umbilical cord and foetal membrane. Stem cells can thus be sorted from such sources, but their localization in each source tissue is not well defined, and cannot be identified in a specific district, isolated from all the cells, more differentiated, which are originated from stem cells.
The low amount of totipotent or multipotent stem cells in human sources different from the embryo requires the use of effective methods of cell sorting/enrichment to make available a sufficient number of stem cells for further applications.
Mesenchimal stem cells, hereafter referred to as MSCs, are adherent, multipotent stem cells that express a panel of surface antigens that is so rich and diversified to limit the possibility to easily distinguish MSCs just on a immuno-phenotypical basis (W. Wagner et al, Experimental Hematology, 33 (2005) 1402-1416).
Methods of “negative” selection, which make use of immunomarkers that label non-MSCs, are employed to deplete these “negative” cells from the total cell populations containing also MSCs. Negative selection, however, do not necessarily assure that, among the depleted population of cells that contain MSCs, are no longer present cells which yet are different from the MSCs. Moreover, negative selection allows neither to sort different MSC subpopulations nor to distinguish MSCs from different sources.
Otherwise, “positive” flow-assisted or magnetic-assisted cell sorting (hereafter FACS, and MACS) methods could induce in the sorted MSCs some physiological suffering (i.e. viability reduction), and they could also influence MSC differentiation capabilities, including unpredictable commitment patterns.
Moreover MSCs can be selected by means of a genetic-engineering approach (Gene Transfer Technology, GTT) that require expensive and time-consuming techniques, high investments for personnel-training and genetic manipulation on cells.
It is, then, still an open issue the possibility to sort/select living, adherent, in particular human totipotent or multipotent stem cells such as the MSCs, by means of a method that is relatively simple, inexpensive, which does not effect cell viability and differentiation ability, and which can be applied by bench personnel of standard specialization.
Human MSCs are actually present in different sources, among which bone marrow, amniotic membrane, chorial membrane, Wharton gel, cord blood and placenta, dental pulp, and lipoaspirates. MSCs from different human sources have shown to exhibit different differentiation potential, likely because of the presence of progenitor cells of different types in tissues of different sources (R. Musina et al., Bulletin of Experimental Biology and Medicine, 141 (2006), 147-151; C. B. Portmann-Lanz et al., Am. J. Obstet. Gynecol., 194 (2006), 662-663). MSCs isolated from the different sources have shown different level for the expression of the pluripotency marker October 4, evaluated by flow cytometry by means of immunostaining.
In these regards, it is important to focus that, though neither morphological nor phenotypical differences have been observed in the human MSCs obtained from different sources (Musina et al., Bulletin of Experimental Biology and Medicine, 139 (2005), 504-509), some preliminary work based on Transmission Electron Microscopy (hereafter referred to as TEM) characterization of the MSCs from different human sources have shown that ultra-structural differences of phenotypically similar MSCs derived from different human tissues can be revelatory of their differences in the in vitro differentiation potential (G. Pasquinelli et al., Ultra-structural Pathology, 31 (1) (2006) 23-31).
These findings have supported, in principle, the idea of using a method for sorting human MSCs of different differentiation potential, which exploits the existence of biophysical differences present in the MSC populations.
Field-flow fractionation (hereafter referred to as FFF) methods are able to distinguish morphological and biophysical differences within cellular populations (A. Lucas et al. In “Field-Flow Fractionation Handbook”; Schimpf, M. E.; Caldwell, K.; Giddings, J. C., Eds.; Wiley-Interscience: New York, 2000; Chapter 29).
The most classical FFF method using the Earth gravity field (hereafter referred to as GrFFF) is disclosed in U.S. Pat. No. 4,214,982.
The hyperlayer mode is disclosed in U.S. Pat. No. 4,830,756 as particularly suited for the rapid fractionation of ultra-high molecular weight polymer. The effectiveness of the method is explained by the application of an entropic force acting on polymeric chain, with the consequence of the fine settlement of separation, with a precise dependence on molecular weight. A mention to the application of the method neither for cell separation nor for adherent cell sorting is reported in this patent.
In the hyperlayer mode GrFFF is able to fractionate different type of cells, among which winemaking yeast cells in suspension, that is not adherent cells, in order to evaluate cell viability for oenologic applications (patent application ES2239886). The method described in this patent application does not concern the separation and characterization of a specific cell subpopulation, but it only proposes the separation of not-adherent, winemaking yeast cells from non cellular material followed by cell viability determination.
Moreover, GrFFF in the hyperlayer mode has shown to be able to fractionate paucipotent stem cells (hemapoietic) from mouse bone marrow. Nevertheless, they are neither adherent nor stem cells at high potency (Urbánková, E. et al.; J. Chromatogr. B, 687 (1996), 449-452). In this work there is no suggestion on the possible employment of GrFFF for the fractionation of cells with morphologic modifications obtained in dynamic conditions; that is that when adherent cells are resuspended they change their conformation, but they reacquire the native conformation with full maintenance of viability once they are back in adhesion.
The inventors have also shown GrFFF as able to fractionate CD34+, human hemapoietic precursor cells, which however are non-adherent cells (12th International Symposium on FFF—Aug. 28-30, 2005; Brno). First indications on the possibility of GrFFF of MSCs from human foetal membrane was also therein given, which however just suggested the possibility to observe qualitative differences between two different samples of MSCs. Neither indications on the possibility to sort the MSCs based on their biophysical differences, nor any evaluation of the so-obtained fractionation recovery and throughput have however been reported.
In fact, GrFFF appears to be unsuitable to high-yield sorting of adherent stem cells such as the MSCs. This is for two fundamental reasons. First, high-through (i.e. preparative-scale) separation methods are know to perform under non-equilibrium conditions. GrFFF in fact develops separation in equilibrium conditions between flow and the applied field. Second, in order to reach such an equilibrium condition necessary to the fractionation mechanism, the GrFFF method requires that, before the flow-assisted fractionation, the cell sample sediments in correspondence of the channel wall of lower gravitational potential (said the “accumulation wall”). This process, said “sample relaxation”, requires that the cell sample is injected at a low flow rate regime, and it usually requires also that the injection flow is stopped to make the cell sample sediment at the accumulation wall.
Sample relaxation then is a necessary condition for the GrFFF mechanism. It can however cause cell-cell aggregation/stacking, and possible cell adhesion to the accumulation wall of the fractionation channel.
Therefore, GrFFF should be advised against its use as a high-throughput sorting technique for adherent stem cells, because it performs under equilibrium conditions, and because the achievement of such equilibrium conditions induce possible cell damaging and viability reduction due to cell-cell and cell-channel interaction, which affect their further usage.
A severe prejudice can be then derived against the use of GrFFF as a high-throughput sorting method for the use of adherent stem cells. This prejudice induce a severe conceptual hindrance to the applicability of GrFFF, particularly in the cases of therapeutic applications of human stem cells, for instance in regenerative medicine applications.
Alternatively, it has been shown that it is possible to perform sample relaxation, however for non adherent cells, without any change on flow rate, by means of sample injection directly on accumulation wall.
A combined technique derived from GrFFF, which uses a dielectrophoretic potential (DEP) in combination with the Earth gravity field, is disclosed in WO0196025, and hereafter referred to as GrFFF/DEP. It was applied to sort also adherent cells such as neoplastic cells, but the application to adherent stem cells and the evaluation of possible effects of the DEP on viability and physiology of stem cells have not been reported. Compared to GrFFF, moreover, GrFFF/DEP requires a more complicated instrumentation, and it shows limitations if a scale-up to a high-throughput format is sought, as in the aims of the present invention. This is because also GrFFF/DEP requires the onset of equilibrium conditions between the Earth gravity field and the DEP
Due to the more intense field than gravitational field, the FFF variant which employs sedimentation field (SdFFF—Sedimentation Field-Flow Fractionation) is able to perform sample relaxation with reduced interactions with accumulation wall, making it useful also for the fractionation of adherent cells.
The SdFFF in hyperlayer mode has shown to be able to fractionate adherent mouse stem cells lines from cell culture for further applications (Guglielmi, L., et al.; Anal. Chem. 76 (2004), 1580-1585). The authors underline that there are critical parameters to be evaluated to obtain the fractionation and enrichment of an enough amount of stem cells for further applications, for the preparation of trans-genic mice and, in particular, with viability maintenance of adherent cells. Some of the authors have already demonstrated that SdFFF operating in the hyperlayer mode is able to separate cells in sterile conditions with fully maintenance of cell viability (Battu S., et al.; J. Chromatogr. B, 751 (2001) 131-141).
A properly designed channel for SdFFF, which can be easy disassembled for carrying out easy cleaning and sterilization/decontamination procedures, and which avoids its deformation during usage was proposed to improve fractionation reproducibility of living cells (US 2006/0151403A1). However, with respect to the GrFFF instrumentation, the SdFFF instrumentation setup is intrinsically more complex. High investments for operation and relative instruments do not allow for a disposable use, which is indeed possible with GrFFF, and which is particularly suited in clinical field. Possible online detection of cells, and maintenance of physiological and sterility conditions during fractionation is also more complicated in SdFFF than in GrFFF. Finally, being like GrFFF a method operating in equilibrium conditions, in principle SdFFF is not able to fractionate a number of cells compatible with those needed for direct medical applications (as in transplants).
Although theoretical different from FFF, the split-flow fractionation with a thin capillary channel (SPLITT—Split Flow Thin Cell) is able to give a high throughput sorting of different cells without relaxation, and it suitable for adherent cells. In specific, the gravity-driven SPLITT technique has been shown able to yield a high throughput binary separation in biocompatible conditions of cell populations characterized by pronounced differences in the average dimensions (Benincasa, M. A. et al; Anal. Chem., 77 (2005) 5294-5301). Nevertheless, the SPLITT technique gives only a binary separation into two cell subpopulations, and it is not able to fractionate cells during their elution on a basis of small biophysical differences, both among cells from a heterogeneous population and among cells recovered from different sources. On the other hand, these features are fundamental in order to fractionate stem cells from more differentiated cells present in the same population or to characterize and select stem cell populations recovered from different sources.